The process of protein synthesis on the ribosome is among the oldest of biological mechanisms, having evolved probably in the RNA world before the first cells. The ribosome has evolved an elaborate structure, the essential parts conserved in all domains of life. Understanding the mechanism by which the ribosome faithfully translates mRNAs into proteins has become both more important and more tractable with the solution of molecular structures of both subunits of bacterial ribosomes and a lower resolution structure of the yeast ribosome. Our laboratory for many years has been interested in the problem of how mRNA sequences interact with the ribosome to cause programmed errors. These errors include programmed translational frameshifts, which target elements of the ribosome involved in maintaining fidelity in order to cause the ribosome to shift reading frames at frequencies approaching 100%. Sine e frameshifting at random sites occurs at much less than one in 20,000 codons, the local change in fidelity is truly immense. Accuracy in protein synthesis consists of accepting the correct (cognate) aminoacyl-tRNAs bound to elongation factor 1A (EF-1A) into the ribosomal A site and rejecting incorrect (non-cognate) ones. Discrimination involves both kinetic proofreading and induced fit in which cognate aa-tRNAs increase the probability of their own acceptance by inducing a structural change in the ribosome. We do not understand the nature of this structural change or its regulation. Venki Ramakrishnan has proposed that part of the mechanism involves destabilizing a protein-protein interaction between ribosomal proteins S4 (rpS4) and rpS5. Breaking this interaction may cause the ribosome to "close", trapping the tRNA in the A site and allowing exit of EF-1A-GDP. The evidence for the model consists of a small number of error-prone mutations that alter residues in and near the interface. We propose to test this hypothesis more stringently by identifying more erro-prone mutations both in the rpS4-rpS5 interface and in a potential interaction pathway in the ribosome leading from the decoding sites. This work will be done both in bacteria and in yeast. We will also study which trans-acting proteins interact with this accuracy mechanism by studying the accuracy effect of a class of candidate gene deletions in yeast.